通过聚合酶链反应扩增及微孔板中的捕获杂交检测1型人类免疫缺陷病毒DNA
Detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtiter wells.
作者信息
Keller G H, Huang D P, Manak M M
机构信息
Cambridge Biotech Corporation, Rockville, Maryland 20850.
出版信息
J Clin Microbiol. 1991 Mar;29(3):638-41. doi: 10.1128/jcm.29.3.638-641.1991.
Abstract
We developed an improved microtiter-based assay for the detection of polymerase chain reaction (PCR)-amplified DNA sequences. The synthetic DNA sequences used to prime the PCR were labeled with biotin at their 5' ends so that the specific PCR product was labeled with biotin. Following amplification, an aliquot of the PCR product was denatured and hybridized to a capture DNA sequence immobilized in a microtiter well. The capture sequence was complementary to a portion of the sequence between the primers, so that only extended primers were captured. The captured PCR product was detected colorimetrically by using a streptavidin-peroxidase conjugate and tetramethylbenzidine substrate.
摘要
我们开发了一种改进的基于微量滴定板的检测方法，用于检测聚合酶链反应（PCR）扩增的DNA序列。用于引发PCR的合成DNA序列在其5'端用生物素标记，以便特异性PCR产物用生物素标记。扩增后，取一份PCR产物进行变性，并与固定在微量滴定孔中的捕获DNA序列杂交。捕获序列与引物之间的部分序列互补，因此只有延伸的引物被捕获。使用链霉亲和素-过氧化物酶共轭物和四甲基联苯胺底物通过比色法检测捕获的PCR产物。
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